Methods for the cultivation of cells and cell biomass in a filter cake

ABSTRACT

Provided herein are filter cake-based systems and methods for cultivating cells and cell biomass therefrom. Provided herein is a system for cultivating cells and cell biomass comprising a filter chamber comprising at least one inlet and at least one outlet, at least one filter support located within the filter chamber, and a filter cake located on the filter support, wherein the filter cake comprises at least one filter aid and a plurality of cells. Provided herein is a method for optimizing the cultivation of cells and cell biomass, comprising providing a filter support, adding at least one filter aid to the filter support, adding a plurality of cells to the filter aid, wherein the cells and the filter aid together comprise a filter cake, growing the cells into a cell biomass in the filter cake, wherein the filter cake is at least partially compressible.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Application No.17/481,176, filed on Sep. 21, 2021. The aforementioned application ishereby incorporated by reference in its entirety.

BACKGROUND

Bringing cell-based meat produced by culturing metazoan cells to themarketplace requires overcoming several hurdles. For example, thecultivation of thick cell-based meat products at large scale can beimpeded by inefficient and suboptimal culturing of cells and tissues. Asa result, the harvested yield and quality may be reduced, necessitatingthe addition of synthetic ingredients, resulting in an increase in costand presence of undesirable additives.

Provided herein are systems and methods for the optimal cultivation ofcell-based meat, as well as other tissue-based applications.

SUMMARY

Provided herein are filter cake-based systems and methods forcultivation of cells and cell biomass.

Some embodiments disclosed herein provide a method for optimizing thecultivation of cells and cell biomass, the method comprising providing afilter support, adding at least one filter aid to the filter support,adding a plurality of cells to the filter aid, wherein the cells and thefilter aid together comprise a filter cake, growing the cells into acell biomass in the filter cake, wherein the filter cake is at leastpartially compressible. In some embodiments, the cell biomass is a meatproduct.

In some embodiments, the method comprises compressing or decompressingthe filter cake by varying one or more of flow rate and pressure. Insome embodiments, the filter cake is edible. In some embodiments, thefilter aid is edible or degradable. In some embodiments, the filter aidcomprises at least one compressible filter aid and at least onenon-compressible filter aid. In some embodiments, the filter aid is atleast partially compressible.

In some embodiments, the filter aid comprises organic fibers of a lengthof between about 1 µm - about 20 µm, about 20 µm - about 50 µm, about 50µm - about 80 µm, about 80 µm - about 110 µm, about 110 µm - about 140µm, about 140 µm - about 170 µm, about 170 µm - about 200 µm, about 200µm - about 230 µm, about 230 µm - about 260 µm, about 260 µm - about 290µm, or about 290 µm - about 320 µm. In some embodiments, the filter aidcomprises organic fiber with a fiver titer of between about 0.01 dtex -about 120 dtex, about 0.05 dtex - about 60 dtex, about 0.1 dtex -about30 dtex, about 0.2 dtex - about 15 dtex, or about 0.5 dtex - about 5dtex. In some embodiments, the filter aid is added to the filter supportat between about 25 g/m² - about 12000 g/m², about 50 g/m² - about 6000g/m², about 100 g/m² - about 3000 g/m², about 200 g/m² - about 1500g/m², or about 400 g/m² - about 750 g/m².

In some embodiments, the filter support is comprised in a horizontalfilter, vertical filter, plate filter, plate press filter, mash filter,cloth filter, mass filter or candle filter. In some embodiments, thetemperature is maintained at between about 10° C. to about 45° C. duringgrowth. In some embodiments, the cells are from a species of poultry,game, aquatic or livestock.

Provided herein are methods for establishing perfusion through a growingcell biomass. In some embodiments, the method comprises seeding cells onan at least partially compressible filter aid, compressing the cells,the at least partially compressible filter aid, or both, flowing mediathrough the at least partially compressible filter aid to grow the cellsinto a cell biomass, whereby growth reduces perfusion over time,decompressing the cells, the at least partially compressible filter aid,or both, to increase perfusion of media. In some embodiments, the cellbiomass is compressed by a fluid pressure or flow of the media. In someembodiments, the cell culture media provides nutrients and oxygenationto promote cell growth. In some embodiments, the cell biomass isdecompressed with a relief valve, a lower pressure media flow, flowreduction of the media, or some combination thereof. In someembodiments, the cell biomass, its substrate, or both are decompressedproportionally to the growth of the cell mass, whereby perfusion isstabilized. In some embodiments, the cell biomass, the at leastpartially compressible filter aid, or both cycle between a compressedstate and a decompressed state at a pulsing frequency. In someembodiments, the pulsing frequency is between about 20 seconds to about20 minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts an illustrative schematic of an exemplary system andmethod of the disclosure for cultivating cells. The exemplary systemshown in FIG. 1A includes a filter chamber containing filter supports influidic communication with two filter aid chambers, containing exemplaryfibrous plant-based filter aids (starch and cellulose), and two celltanks containing exemplary cells (myoblasts and fibroblasts),respectively.

FIG. 1B depicts an exemplary embodiment of adding a filter aid to filtersupports to form pre-coat layers.

FIG. 1C depicts an exemplary embodiment of adding cells and/or filteraids on to filter supports to form filter cakes.

FIG. 1D depicts another exemplary embodiment of adding cells and/orfilter aids on to filter supports to form filter cakes.

FIG. 1E depicts an exemplary embodiment of compressing and decompressingfilter cakes by pressure and/or flow variations.

FIGS. 2A-2N depict another exemplary system and method of the disclosurefor cultivating cells and cell biomass.

FIGS. 3A-3M depict yet another exemplary system and method of thedisclosure for cultivating cells and cell biomass.

DETAILED DESCRIPTION

Before describing particular embodiments in detail, it is to beunderstood that the disclosure is not limited to the particularembodiments described herein, which can vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular illustrative embodiments only and is not intendedto be limiting unless otherwise defined. The terms used in thisspecification generally have their ordinary meaning in the art, withinthe context of this disclosure and in the specific context where eachterm is used. Certain terms are discussed below or elsewhere in thespecification, to provide additional guidance to the practitioner indescribing the compositions and methods of the disclosure and how tomake and use them. The scope and meaning of any use of a term will beapparent from the specific context in which the term is used. As such,the definitions set forth herein are intended to provide illustrativeguidance in ascertaining particular embodiments of the disclosure,without limitation to particular compositions or biological systems.

Standard techniques may be used for recombinant DNA, tissue culture, andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques may be performed according to manufacturer’sspecifications or as commonly accomplished in the art or as describedherein. These and related techniques and procedures may be generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. Unless specificdefinitions are provided, the nomenclature utilized in connection with,and the laboratory procedures and techniques of, molecular biology,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well-known andcommonly used in the art. Standard techniques may be used forrecombinant technology, molecular biological, microbiological, chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery.

As used in the present disclosure and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

Throughout the present disclosure and the appended claims, unless thecontext requires otherwise, the word “comprise,” or embodiments such as“comprises” or “comprising,” will be understood to imply the inclusionof a stated element or group of elements but not the exclusion of anyother element or group of elements.

As used in the present disclosure the term “about” shall generally meanan acceptable degree of error for the quantity measured. Exemplarydegrees of error are within 20%, within 10%, and within 5% of a value orrange of values.

Cultivation of cells dispersed in suspension typically limits theirassembly into higher order structures, such as tissues and organs. Thus,their use may not be optimal for several applications, including theproduction of cell-based meat, cultivation of stem cells, liver cells,cell spheroids, nodules, organoids, artificial organs, 3D cell culture,bio-fabrication, regenerative bio-engineering, and the like. Cells grownin suspension do not typically form significant interconnections witheach other. The few smaller cell-clusters or pellets that do form maynot grow significantly and may be unable to fuse and form cross-linkedmulti-nuclei higher-order tissue assemblies, such as muscle fibers. Asthis is important for the large-scale manufacturing and consistency ofcell sheets, inclusive of cell-based meat products, the cells are eitherseparated in further processing steps (e.g. centrifugation) and mixedwith other substances to a semi-finished or finished product, or areadhered on to a surface, (e.g. by sedimentation) to form thin tissuelayers. However, both these methods are often suboptimal andinefficient. If the individual cells are separated without significanttissue formation, additives (such as enzymes and/or fibers) aretypically used for texturing, which remain at least partially in thecell-based meat, raising quality, batch-to-batch variability, and safetyconcerns. Further, since the cells are no longer actively supplied withoxygen and nutrients under these conditions, they may quickly lyse,inhibiting any further increase in cell biomass or even cell fusion. Ifthe cells are adhered to a surface, a tissue layer formed bycross-linking, cell fusion and cell biomass increase may be formed.However, because the growing tissue increasingly hinders the diffusionof nutrients and oxygen into deeper layers, the viable tissue thicknessis severely limited, and often results in lysing of cells in the lowerboundary layers towards the supporting structure. This limitation ofnon-homogeneous cell and tissue culture is also seen with cultivationprocedures using matrix structures, such scaffolds, to increase thesurface area of adhesion. Provided herein are systems and methods forcultivation of cells that overcome the above-mentioned drawbacks.

Described herein are systems and methods in which cells in suspensionare added and cultured using filtration in a filter cake. As usedherein, a filter cake comprises a plurality of cells and at least onefilter aid. The filter cake may be located on a filter support. Withoutbeing bound to theory or mechanism, in some embodiments, the filtrationflow guides the free-floating dispersed suspension cells to a compactfilter cake for cultivation. The flowing media allows for perfusion ofthe cells in the filter cake, providing essential nutrients and oxygenunder sterile conditions, enabling cell growth, cell division, cellfusion, and cross-linking. Cellular waste products are also activelyeliminated by the flow. Adequate and homogeneous supply of growthessential nutrients and gases reaching even the growing cells in deeperlayers. Thus, the cells in the filter cake form thick robust tissues andhigher order cell biomass assemblies (e.g. fusion of myoblasts intomulti-nucleated myotubes). Properties of the filter cake, such asporosity, permeability, compressibility, can be individually tunedaccording to the specific needs of the cells/cell biomass, by adjustingparameters such as the flow volume per time, the flow direction, and/orthe pressure. Mixtures of different cell types and/or filter aids can beused to create specific structural and process influencing features inthe filter cake, thus, overall efficiency of producing cell-based meatis dramatically improved, bringing down costs

These systems and methods are applicable not only for the production ofcell-based meat, but any other applicable field, such as therapeutics,bio-manufacturing, bio-engineering, cultivation of stem cells, livercells, cell spheroids, nodules, organoids, artificial organs, 3D cellculture, bio-fabrication, regenerative bio-engineering, and the like.

Filter Cake-Based Systems for Cultivating Cells and Cell Biomass

Provided herein are filter cake-based systems for cultivating cells andcell biomass. In some embodiments, the system for cultivating cellscomprises a filter chamber comprising at least one inlet and at leastone outlet, at least one filter support located within the filterchamber, and a filter cake located on the filter support, wherein thefilter cake comprises at least one filter aid and a plurality of cells.

FIG. 1A shows an exemplary system 100 of the present disclosure. System100 may comprise a filter chamber 110 with an inlet 112, two outlets 114and 116, and a bypass (B) outlet 118. The filter chamber may be dividedby the filter support into unfiltered (also referred to as unfiltrate ornon-filtered) chamber 192 and filtered (also referred to as filtrate)chamber 194. In this exemplary system, two filter supports, 120 and 122may be located within the filter chamber 110. In alternativeembodiments, the filter chamber may include as many as hundreds offilter supports to support substantial and robust cell growth. Filteraids 132 and 142 may be stored in the filter aid chambers 130 and 140,respectively. In some embodiments the filter aid may be a plant-basedfiber. In some embodiments, the filter aid 132 may be (native) starch(S) and 142 may be cellulose (C). Cells, such as myoblasts andfibroblasts, may be stored in one or more cell tanks (150 and 160). Asshown in FIG. 1A, myoblasts (M) 152 and fibroblasts (F) 162 may bestored in the cell tanks 150 and 160, respectively. The filter aidchambers 130 and 140, the cell tanks 150 and 160, and the filter chamber110 may be in fluidic communication with each other. A heat exchanger170 (H) may be used to regulate the temperature of the filter chamber.As shown in FIG. 1D, filter cakes 126 and 128, comprised of the filteraids (starch (132) and cellulose (142)), and cells (myoblasts (152) andfibroblasts (162)) may form on the filter supports.

Cells and Cell Biomass

The cells and cell biomass thereof used in the systems and methodsdescribed herein may be produced by the in vitro culturing of naturallyoccurring, genetically engineered, or otherwise modified cells inculture.

The systems and methods provided herein are applicable to any metazoancell in culture. In some embodiments, in the context of using thesystems and methods of the disclosure to generate cell-based meat, thecells are from any metazoan species whose tissues are suitable fordietary consumption. In some embodiments, the cells may demonstrate acapacity for differentiation into mature tissue, such as skeletal muscletissue, other muscle tissues, or any cell, cellular biomass, and/ortissue that can be consumed as cell-based meat or nutrients thereof. Thecells in the present disclosure may be primary cells, or cell lines. Thecells may be adherent-cells or non-adherent cells, flocculating ornon-flocculating cells.

In some embodiments, the cells are derived from any non-human animalspecies intended for human or non-human dietary consumption (e.g. cellsof avian, ovine, caprine, porcine, bovine, piscine origin),cells oflivestock, poultry game, or aquatic species, etc.).

In some embodiments, the cells are from livestock such as domesticcattle, pigs, sheep, goats, camels, water buffalo, rabbits, and thelike. In some embodiments, the cells are from poultry such as domesticchicken, turkeys, ducks, geese, pigeons, and the like. In someembodiments, the cells are from game species such as wild deer,gallinaceous fowl, waterfowl, hare, and the like. In some embodiments,the cells are from aquatic species or semi-aquatic species harvestedcommercially from wild fisheries or aquaculture operations, or forsport, including certain fish, crustaceans, mollusks, cephalopods,cetaceans, crocodilians, turtles, frogs, and the like.

In some embodiments, the cells are from exotic, conserved, or extinctanimal species. In some embodiments, the cells are from Gallus gallus,Gallus domesticus, Bos taurus, Sous scrofa, Meleagris gallopavo, Anasplatyrynchos, Salmo salar, Thunnus thynnus, Ovis aries, Coturnixcoturnix, Capra aegagrus hircus, or Homarus americanus. Accordingly,exemplary cell-based meat products of the disclosure include avian meatproducts, chicken meat products, duck meat products, and bovine meatproducts.

In some embodiments, the cells are primary stem cells, self-renewingstem cells, embryonic stem cells, pluripotent stem cells, inducedpluripotent stem cells, or differentiated progeny of stem cells.

In some embodiments, the cells are modified to differentiate intoskeletal muscle tissue, connective tissue, fat tissue, and/or any othermature tissue, e.g. useful for cultured meat production or some otherapplication.

In some embodiments, the cells are myogenic cells, programmed to becomemuscle, or muscle-like cells. In some embodiments, the myogenic cellsare natively myogenic, e.g. myoblasts. Natively myogenic cells include,but are not limited to, myoblasts, myocytes, satellite cells, reservecells, side population cells, muscle derived stem cells, mesenchymalstem cells, myogenic pericytes, or mesoangioblasts.

In some embodiments, cells are of the skeletal muscle lineage. Cells ofthe skeletal muscle lineage include myoblasts, myocytes, and skeletalmuscle progenitor cells, also called myogenic progenitors that includesatellite cells, reserve cells, side population cells, muscle derivedstem cells, mesenchymal stem cells, myogenic pericytes, andmesoangioblasts.

In other embodiments, the cells are not natively myogenic (e.g. arenon-myogenic cells such as fibroblasts or non-myogenic stem cells thatare cultured to become myogenic cells in the cultivationinfrastructure).

In some embodiments, the cells of the cellular biomass are somaticcells. In some embodiments, the cells of the cellular biomass are notsomatic cells.

In some embodiments, the cells are genetically edited, modified, oradapted to grow without the need of specific ingredients includingspecific amino acids, carbohydrates, vitamins, inorganic salts, tracemetals, TCA cycle intermediates, lipids, fatty acids, supplementarycompounds, growth factors, adhesion proteins, and recombinant proteins.

In some embodiments, the cells may comprise any combinations of themodifications described herein.

The cell-based meat of the present disclosure, generated using the cellmedia formulations provided herein, is suitable for both human andnon-human consumption. In some embodiments, the cell-based meat issuitable for consumption by non-human animals, such as domesticatedanimals. Accordingly, the cell media formulations provided hereinsupport the growth of “pet food”, e.g. dog food, cat food, and the like.

In some embodiments, the systems and methods may enable production ofthick tissues without the need for an added internal scaffold to supporttissue dimensionality.

In some embodiments, the cells may be cultivated for therapeutic,diagnostic, bio-manufacturing, or bio-engineering applications. Examplesinclude, cultivation of stem cells, liver cells, cell spheroids,nodules, organoids, artificial organs, lab-on-a-chip, 3D cell culture,bio-fabrication, regenerative bio-engineering, and the like.

Filter Support

The systems described in this disclosure comprise at least one filtersupport. In some embodiments the system may comprise a plurality offilter supports. For example, the system may comprise at least 1, 5, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 5000,10,000, 100,000, or 1,000,000 filter supports.

The filter support may be located within a filter chamber. In someembodiments, the system comprises a plurality of filter supports, forexample, the system shown in FIG. 1A contains two filter supports 120and 122 located within the filter chamber 110. The filter supports maybe made of permeable structure like nets, cloughs, mashes, or sinteredmaterials and are oftentimes produced as plates or candles.

In some embodiments the filter support comprises a horizontal filter,vertical filter, plate filter, plate press filter, mash filter, clothfilter, mass filter or a candle filter. In some embodiments, the filtersupport may be contained or installed in horizontal filters, verticalfilters, plate filters, plate press filters, mash filters, clothfilters, or candle filters. Any suitable and sterilizable filter supportknown in the art may be used. A person skilled in the art wouldunderstand how to identify and use a filter support in the systemsdisclosed herein.

The filter support may be of any suitable dimension. In some embodimentsthe filter support has a surface area of between about 1 meter² – about2000 meter².

In some embodiments, the filter support is sterilized before use.

Filter Aid

The systems described in this disclosure comprise at least one filteraid. In some embodiments, the filter aid is stored in filter aidchambers in fluidic communication with the filter chamber. For example,the system shown in FIG. 1A contains two filter aids 132 and 142 storedin filter aid chambers 130 and 140 respectively.

In some embodiments, the filter aid may be composed of incompressiblematerials (also referred to as non-compressible herein), such as starch,diatomaceous earth, perlites, active charcoal and crystals, orcompressible materials, such as vegetable fats, waxes, cellulose,fibers, plant fibers, fungal mycelia, algae, naturally occurring fibers,synthetic fibers, inorganic fibers, organic fibers, wood chips, sprouts,husks, organic shells, plant based proteins, or combinations thereof.Any suitable filter aid or combination of filter aids known in the artmay be used herein. A person skilled in the art would understand how toidentify and use a filter aid in the systems disclosed herein.Combinations of different filter aids with different material propertiessuch as particle sizes, functional surfaces, particle structures, anddifferent compressibility may further be used and combined. For example,combinations of compressible filter aids (e.g. cellulose) andincompressible filter aids (e.g. starch) may be combined at differentratios to form partially compressible filter aids.

The filter aids may be of any suitable length. In some embodiments, thefilter aid may have a length of between about 1 µm - about 20 µm, about20 µm - about 50 µm, about 50 µm - about 80 µm, about 80 µm - about 110µm, about 110 µm - about 140 µm, about 140 µm - about 170 µm, about 170µm - about 200 µm, about 200 µm - about 230 µm, about 230 µm - about 260µm, about 260 µm - about 290 µm, or about 290 µm - about 320 µm. In someembodiments, the filter aid may have a length of no more than about 300µm. In some embodiments, the filter aid may have a length of betweenabout 20 µm - about 50 µm.

In some embodiments, the filter aid may comprise an organic fiber. Insome embodiments, the organic fiber may have a length of between about 1µm - about 20 µm, about 20 µm - about 50 µm, about 50 µm - about 80 µm,about 80 µm - about 110 µm, about 110 µm - about 140 µm, about 140 µm -about 170 µm, about 170 µm - about 200 µm, about 200 µm - about 230 µm,about 230 µm -about 260 µm, about 260 µm - about 290 µm, or about 290µm - about 320 µm. In some embodiments, the organic fiber may have alength of no more than about 300 µm. In some embodiments, the organicfiber may have a length of between about 20 µm - about 50 µm.

In some embodiments, the organic fiber may comprise a fiver titer ofbetween about 0.01 dtex - about 120 dtex, about 0.05 dtex - about 60dtex, about 0.1 dtex - about 30 dtex, about 0.2 dtex - about 15 dtex, orabout 0.5 dtex - about 5 dtex.

In some embodiments, the filter aid may be compressible. In someembodiments, at least one of a plurality of filter aids being used maybe compressible. In some embodiments, at least one component of thefilter aid may be compressible. Non-limiting examples of compressiblefilter aids or their components include: cellulose, organic fibers, andplant fibers.

In other embodiments, the filter aid may be non-compressible orincompressible. In some embodiments, at least one of a plurality offilter aids being used may be non-compressible. In some embodiments, atleast one component of the filter aid may be non-compressible.Non-limiting examples of non-compressible filter aids include:diatomaceous earth, perlites active charcoal, and native starchparticles.

In some embodiments, the filter aid may be partially compressible. Insome embodiments, a single filter aid may be partially compressible byitself. In some embodiments, one or more compressible filter aids andnon-compressible filter aids may be combined to form a partiallycompressible filter aid.

In some embodiments, the filter aid is compressible by between about70% - about 75%, about 75% - about 80%, about 80% - about 85%, about85% - about 90%, about 90% - about 95%, about 95% - about 98%, or about98% - about 100%.

In some embodiments, a combination of at least one compressible filteraid (CFA) and at least one non-compressible filter aid (NCFA) may beused. In some embodiments, the combination of CFA and NCFA may bebetween about 70% CFA and about 30% NCFA, about 75% CFA and about 25%NCFA, about 80% CFA and about 20% NCFA, about 85% CFA and about 15%NCFA, about 90% CFA and about 10% NCFA, about 95% CFA and about 5% NCFA,about 98% CFA and about 2% NCFA, or about 99% CFA and about 1% NCFA. Insome embodiments, the combination of CFA and NCFA comprises at leastabout 98% CFA. In some embodiments, the combination of CFA and NCFA maycontain no more than about 10%, about 9%, about 8%, about 7%, about 6%,about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about0.1%, or about 0.01% NCFA. In some embodiments, the combination of CFAand NCFA may contain no more than about 5% NCFA.

In some embodiments, the combination of a CFA and a NCFA may be a ratioof between about 99CFA:1NCFA to about 1CFA:99NCFA. In some embodiments,the combination of a CFA and a NCFA may be a ratio of about90CFA:10NCFA. In some embodiments, the combination of a CFA and a NCFAmay be a ratio of about 95CFA:5NCFA. In some embodiments, thecombination of a CFA and a NCFA may be a ratio of about 98CFA:2NCFA.

In some embodiments, the ratio of a CFA to NCFA may be between about99:1 to about 1:99. In some embodiments, the ratio of a CFA to NCFA maybe between about 90:10. In some embodiments, the ratio of a CFA to NCFAmay be between about 95:5. In some embodiments, the ratio of a CFA toNCFA may be between about 98:2.

The filter aids may be added in any suitable order. In some embodiments,the filter aids may be added at the same time, sequentially,alternately, or in any relevant order and any time interval.

In some embodiments, the filter aid may be edible. Examples of ediblefilter aids include fungal mycella, plant fibers, and cellulose. The useof edible filter aids avoids additional steps required to remove ordegrade filter aids during the harvesting step, described below.

In some embodiments, the filter aid may be degradable. Examples ofedible filter aids are cellulose, fungal mycella, algae, plant fibers,starch particles, and plant-based proteins.

In some embodiments, the filter aid may be hollow or otherwise shaped,for example to allow for the homogeneous gaseous (e.g. oxygen) andnutrient supply to the cells being cultivated. Besides hollow, orsectioned fibers, the shape could be L, M, Y, Z, square, round, and/orstarshaped in diameters, and straight or cross linked. In someembodiments, the filter aid may be natural or synthetic, in pure form,or with additives, such as coatings. In some embodiments, the filter aidis sterilized before usage.

Filter Chamber

The systems described in this disclosure comprise at least one filterchamber. The filter chamber contains at least one of the filtersupport(s) described above. In some embodiments, the filter chamber maybe divided by the filter support into unfiltered chamber 192, in whichthe unfiltered fluids, cells and filter aids are introduced, andfiltered chamber 194, which is the outlet for the filtered suspensions(FIG. 1A). The filter chamber may have at least one inlet and one outletand may be in fluidic communication with other components of the system.For example, filter chamber 110 shown in FIG. 1A contains the filtersupports 120 and 122, and in fluidic communication via inlet 112 andoutlets 114, 116, and bypass outlet 118. The bypass outlet can ensurethat the filter cake thickness and cell distribution remain homogeneousover the length of the filter support. In one example, the bypass outletrelieves pressure in the unfiltered chamber and the pressure relief mayresult in a decompression or relaxation of the filter cake.

The filter chamber may be of any suitable dimension. In someembodiments, the filter chamber has a size of between about 5 liters -about 25,000 liters. In some embodiments, the filter chamber may be abioreactor.

In some embodiments, the filter chamber may be sterilized. Sterilizationmay be carried out with any known method, using heat, steam, pressure,irradiation, gases, and/or chemicals.

Filter Cake

The systems described in this disclosure comprise at least one filtercake. The filter cake comprises at least one filter aid and a pluralityof cells. In some embodiments, the filter cake is located on the filtersupport. For example, FIG. 1D shows the filter cakes 126 and 128 formedon the filter supports 120 and 122, respectively, in the unfilteredchamber 192.

One of the advantages of the systems disclosed herein is the ability tocustomize the properties of the filter cake. This may be achieved, forexample, by using the appropriate type of filter aid, adding filter aidsadded at the same time as cells, sequentially, alternately, or in anyrelevant order, adding the filter aids accordingly to certain feedbackslike rising differential pressures, and modulating the pressure and flowcharacteristics of the media.

In some embodiments the filter cake is porous. The permeability of thefilter cake can allow for media to actively flow throughout the cake,the cells, and different layers of growing tissue, allowing fornutrients, dissolved oxygen, and growth-relevant substances to penetratedeeply into the filter cake, and undesirable metabolic products to beflushed out. Filter aids with hollow shapes can allow for efficientpenetration, especially to deeper lying tissues. Consequently, it isexpected that the cells are provided a nutrient rich dynamic surface toadhere, grow, cross-link, and form thick layers of tissues.

Based on the type of filter aid used, the filter cake may becompressible or non-compressible. In some embodiments, the filter cakeis compressible. For example, filter cakes containing either onlycompressible filter aids (e.g. cellulose), or compressible filter aidscontaining little or no non-compressible filters aids (e.g. less than5%), in addition to cells, would be considered compressible. Periodicpressure and/or flow induced variations between the inlet and outlet maybe used to compress and decompress the filter cake intentionally. Thistunable compression and decompression of the filter cake is yet anotheradvantage of the systems disclosed herein. This “sponge-like” action,shown by arrows 460 in FIG. 1E for the filter cakes 126 and 128, isexpected to enable deeper infiltration of the media and oxygen into thegrowing layers of cells and tissues, enabling exchange of signalingmolecules, and flushing out waste. In some embodiments, theinterval-like contraction may achieve a desired stimulation of a tissuebeing cultivated, e.g. a muscle or muscle-like tissue. The system maycomprise a flow control pump 500 (FQ) as shown in FIG. 1E, where thefilled in triangle signifies valves and the circle around the filled intriangle signifies a pump. The system may also comprise a nitrogen inlet510 (N) for drying and/or oxygen displacement (FIG. 1E).

In general, the filter cake is a dynamic structure. As culture media isflowed onto it and through it, the various cells begin to grow and maymake connections with neighboring cells, thereby forming a cell biomass.As the cells and the cell biomass grow, the fluid dynamics of theunfiltered chamber to the filtered chamber are in constant flux.Typically, as the cells and the cell biomass grow, the filter cakebecomes more dense and less porous. If the flow rate of cell culturemedia is held constant, despite cell growth, then pressure may build inthe unfiltered chamber, thereby leading to further compression of thefilter cake and a further decrease in porosity. Left unchecked, aconstant flow rate may clog the filter given enough time for robust cellgrowth. In some instances, the flow rate is reduced to maintain aconstant pressure difference between unfiltered chamber and filteredchamber. Such a reduction in flow rate counteracts the resistanceincreases that result from increasing filter cake thickness. In someembodiments, a computer is configured to control a flow pump and read anunfiltered chamber pressure sensor, whereby the computer can establishand maintain a stable pressure differential between the unfilteredchamber and filtered chamber by taking pressure sensor readings andmodulating the flow pump.

In some embodiments, the pressure differential between the unfilteredchamber and filtered chamber are intentionally varied and this variancemay be carried out in a repeated or rhythmic fashion. In one example,the filter cake may be periodically compressed and decompressed tomaintain the porosity of the filter cake and/or to press the mediumsufficiently into the deeper filter cake layers. In this example, thefilter cake may exhibit mechanical properties similar to a sponge underthe varied pressure. As the pressure is increased, the filter cake iscompressed, cells aggregate, porosity decreases, cells are pushed deeperinto the filter aids, and cell culture media is ejected through thefilter support into the filtered chamber. As the pressure is decreased,the filter cake is decompressed, space opens up around the aggregatedcells (e.g. cell mass), porosity increases (thereby allowing cellculture media to perfuse to cells deep in the filter cake), and flowthrough the filter cake is increased. The compression engagement may becharacterized by a rate at which pressure builds and a duration forwhich a high pressure state is maintained. Likewise, the decompressionengagement may be characterized by a rate at which pressure decreasesand a duration for which a low pressure state is maintained. The flow ofthe media may be manipulated to oscillate the filter cake between highpressure and low pressure states at a frequency in a seconds range, aminutes range, an hours range, or in a days range. In an exemplaryembodiment manipulating the pressure differential between the unfilteredchamber and filtered chamber enhances perfusion to cells deep within thefilter cake and mitigates the risk of cell death from lack of oxygen ornutrients. In some instances, pressure increases counteract the filtercake’s increased resistance to perfusion caused by the growing andexpanding cell mass by compressing the cells together, whereby asubsequent pressure decrease results in extra open space around therecently compressed cells where fluid can then freely perfuse.

In some embodiments, the filter cake may be compressed by pressurevariations. Any suitable pressure range may be used. In someembodiments, the pressure between the inlet and the outlet is betweenabout 0.01 bar - about 500 bar, about 0.05 bar - about 50 bar, or about0.1 bar -about 5.0 bar. In some embodiments, the pressure between theinlet and the outlet is between about 0.1 - about 5.0 bar.

In some embodiments, the filter cake may be compressed by flow ratevariations. Any suitable flow rate may be used. In some embodiments, theflow rate between the inlet and the outlet is between about 0.01 L/min -about 100,000 L/min, about 0.1 L/min - about 10,000 L/min, or about 1L/min - about 1000 L/min. In some embodiments, the flow rate between theinlet and the outlet is between about 10 L/min - about 1000 L/min.

Another advantage of the systems disclosed herein is the ability tocultivate multiple cell types, in any desired order. For example, thefilter cake may contain multiple layers of cell biomass, wherein eachlayer is of a different cell type, such as alternating layers ofmyoblasts, adipocytes and fibroblasts. In some embodiments, the filtercake may contain one or more cell types. In some embodiments the filtercake may contain one or more filter aids added in any relevant order. Insome embodiments, the filter cake may contain two, three, or more celltypes. The plurality of cell types may be added in any relevant order,such as in alternating layers. The filter cake may comprise multiplelayers of different cell types and different filter aids.

In some embodiments, the filter cake may contain skeletal muscle cells,stem cells, pluripotent stem cells, embryonic stem cells, inducedpluripotent stem cells, fibroblasts, myoblasts, somatic cells,extraembryonic cells, myocytes, satellite cells, side population cells,muscle derived stem cells, mesenchymal stem cells, myogenic pericytes,mesoangioblasts, and adipocytes. In some embodiments, the filter cakemay contain co-cultures of fibroblast and myoblasts at a ratio ofbetween about 95F:5M to about 5F:95M. In some embodiments, the filtercake may contain cells from a species of poultry, game, aquatic orlivestock. In some embodiments the filter cake may contain cells fromGallus gallus, Meleagris gallopavo, Anas platyrhynchos, Bos taurus, Susscrofa, Ovis aries, Salmo salar, Thunnus thynnus, Gadus morhua, Homarusamericanus, Litopenaeus setiferus, Oncorhynchus mykiss, or Oreochromisniloticus.

Any suitable density of cells may be cultivated in the filter cake. Insome embodiments, the initial density of cells in the unfiltered mediumis between about 1 - 200x10⁶ cells/ml. In some embodiments, the densityof cells in the medium is between about 0.05x10⁶ cells/ml - about2000x10⁶ cells/ml, about 0.5x10⁶ cells/ml - about 200x10⁶ cells/ml,about 5x10⁶ cells/ml - about 20x10⁶ cells/ml, or about 5x10⁶ cells/ml -about 10x10⁶ cells/ml.

In some embodiments, the filter cake is edible, meltable, and/ordegradable.

Heating Element

The systems described in this disclosure may be heated. In someembodiments, the system may comprise at least one heating element. Anysuitable heating element known in the art may be used. A person skilledin the art would understand how to identify and use a heating element inthe systems disclosed herein. In some embodiments, the heat element maybe a heat exchanger, like a tubular or plate heat exchanger. Forexample, the system shown in FIG. 1A contains an in-line heat exchanger170. In some embodiments, the system may be placed in atemperature-controlled environment.

The heating element may be located at any one or more components of thesystem, such as the filter chamber, filter aid chambers, cell tanks,inlets, outlets, within the transfer piping etc. In some embodiments,the heating element 170 is connected to the filter chamber 110, as shownin FIG. 1A. In some embodiments, the heating element may be connected tothe filter aid chambers 130 and 140. In some embodiments, the heatingelement may be connected to the cell tanks 150 and 160. In someembodiments, the heating element may be connected to any one or more ofinlet 112, outlets 114, 116, and 118, or any other component (e.g.pipes, valves) of the system used for fluidic communication. Othermethods of heating, such as jacketed tanks or temperature-controlledenvironments would be well known to those skilled in the art.

The heating element may maintain any suitable temperature required inthe system. In some embodiments, the heating element may regulate atemperature suitable for cell cultivation. In some embodiments, theheating element may regulate the temperature of the filter chamberbetween about 10° C. – about 45° C. during growth. In some embodiments,the heating element may regulate a temperature of between about 37° C.In some embodiments, the heating element may regulate a temperatureappropriate for sterilization, such as about 100° C. or above.

In some embodiments, the heating element may regulate a temperatureappropriate for storing and/or sterilizing cell culture media, nutrientmedia, buffers, saline, or any other liquids.

In some embodiments, the heating element may regulate the temperature ofthe cells or cell biomass produced therefrom. In some embodiments, theheating element may regulate the temperature of the cell-based meat,and/or wash buffers. In some embodiments, the temperature differencebetween different steps (such as after pre-coat layer formation andbefore adding cell suspensions) of the method does not exceed aparticular temperature (e.g. about 5° C.) to avoid damaging the cells.In some embodiments, before harvesting, the cell-based meat may becooled down for further processing. In some embodiments, the cell-basedmeat may be heated for a certain period of time. In some embodiments,the pre-coating of the filter support with a first filter aid can bedone at a high temperature to ensure sterility, before introducingcooler media to introduce cells into the process.

Media

Different types of media may be used depending on the use in the systemand methods. The media described herein may have any appropriate sugar,salt, amino acids, vitamins, preservatives, buffers, osmolarity, pHvalue, temperature, ionic strength, viscosity, and/or ingredients (e.g.other nutrients, signaling molecules). The combination of these factorswill depend on the type and application of the media, as describedbelow. In some embodiments, the media disclosed herein may be sterile.

In some embodiments, the media is a flooding media. Flooding media maybe used to add one or more filter aids on to the filter chamber to forma uniform layer of the filter aid (also referred to herein as pre-coatlayer). For example, as shown in FIG. 1B the flooding media (FM) 180 maybe used to add the filter aid starch 132 on to the filter supports toform the pre-coat layers 124 and 125, respectively. Examples of floodingmedia include sterile cell culture medium, water, and/or sterile saline.The flooding media may be heated for sterilization.

In some embodiments, the media is a filter aid media. The filter aidmedia may be used to suspend one or more of the filter aids. Examples offilter aid media include water, salted liquids, saline, minerals andmetals, PBS, HEPES, or other media known to a person of skill in theart, to achieve, for example, the desired osmolarity, density, andfunctionality. For example, as shown in FIG. 1A, the filter aids 132(starch) and 142 (cellulose) are suspended in saline as the filter aidmedia. Depending on the type of filter aid used, such asnon-compressible starch or compressible cellulose, different filter aidmedia may be used. The filter aid media containing the filter aids maybe heated for sterilization or cooled to prevent degradation. The filteraid media may be homogenized using an agitator, a circulation pumpand/or a gas-sparger to prevent settling or floating of the filter aids.A gasification may also be implemented to prevent or force oxidation.

In some embodiments, the media is a cell culture media. The cell culturemedia may be used to suspend cells for cultivation. Examples of cellculture media include Eagle’s Minimum Essential Media (EMEM), Dulbecco’smodified Eagle’s Medium (DMEM), RPMI-1640, F12 Medium, Ham’s nutrientmixtures, and other nutrient media known to a person of skill in theart. In some embodiments, the media is chemically defined. In someembodiments, the media is free of animal derived serum. In someembodiments, the media is free of animal derived components.

In some embodiments, the media is a wash-out media. In some embodiments,the cell biomass is a cell-based meat. The wash out media may be used toharvest the cell-based meat and/or to impart the physical and/ororganoleptic properties to the cell-based meat. Different wash out mediamay be used depending on the type of cell-based meat and the desiredsensory and visual characteristics. For example, aqueous solutionscontaining red beet juice may be used for a red coloring of thecell-based meat, or other components such as iron may be added forflavor.

In some embodiments, the different media used herein have the same orsimilar osmolarity, to ensure that the cells do not suffer osmoticstress. In some embodiments, the filter aid media and the cell culturemedia containing the cells have the same or low osmotic difference. Inother embodiments, the osmotic pressure might intentionally differsignificantly in some parameters, to remove undesired substances out ofthe product by diffusion or introduce desired substances into theproduct, which improve the products characteristics.

In some embodiments, any of the above media may be reused afterfiltration. For example, the media collected through outlet (R) 190(FIG. 1C) from the filtered chamber, may be fully or partially reused,recirculated, or enriched as required with nutrients, oxygen, buffers,signaling molecules, etc.

In some embodiments, the media may contain substances which preventmicrobial growth. In other embodiments, it also includes substancesspecifically chosen to improve the final cell biomass’s shelf life,chemical and/or physical stability, flavor, texture, color, odor, orsome combination thereof.

Filter-Cake Based Methods for Cultivating Cells and Cell Biomass

Provided herein are methods for optimizing the cultivation of cells andcell biomass. In some embodiments, the method comprises, providing afilter support, adding at least one filter aid to the filter support,adding a plurality of cells to the filter aid, wherein the cells and thefilter aid together comprise a filter cake, and growing the cells into acell biomass in the filter cake, wherein the filter cake is at leastpartially compressible.

Providing a Filter Support

The methods disclosed herein may include providing at least one filtersupport. Any suitable filter support may be used, as described in theabove section. Examples of filter supports include: nets, mashes, grits,cloth, sintered, porous, and perforated materials usually installed intohorizontal filters, vertical filters, plate filters, plate pressfilters, mash filters, cloth filter or candle filters. The filtersupport may be of any suitable dimension. In some embodiments, thefilter support has a surface area of between about 1 meter² – about 500meter².In some embodiments the filter support has a surface area ofbetween about 1 meter² – about 2000 meter².

In some embodiments, the at least one filter support may be locatedwithin a filter chamber. For example, FIG. 1A contains two filtersupports 120 and 122 located within the filter chamber 110. In someembodiments, the filter support may be sterilized before being placedwithin the filter chamber. The filter chamber may be emptied, cleaned,and sterilized before placing the filter support. In some embodiments,the filter support and chamber are sterilized together.

Adding Filter Aids

The methods disclosed herein include adding at least one filter aid tothe filter support. Any suitable filter aid may be used, as describedabove. Examples of filter supports include cellulose, starch,diatomaceous earth, perlites, active charcoal, vegetable fats, waxes,crystals, fibers, plant fibers, fungal mycelia, algae, naturallyoccurring fibers, synthetic fibers, inorganic fibers, organic fibers,husks, plant based proteins, fats, waxes, or combinations thereof.Combinations of different filter aids with different material propertiessuch as particle sizes, functional surfaces, particle structures, anddifferent compressibility (e.g. compressible or incompressible) may beused.

Adding the filter aid to the filter support may be carried out by anysuitable method. In some embodiments, the at least one filter aid may beadded by flowing media containing the at least one filter aid throughoutthe filter camber. The filter aid may be suspended in a fluid and addedonto the filter support. For example, as shown in FIG. 1B, the filteraids starch 132 and cellulose 142, are suspended in the filter aidmedium saline, and stored in filter aid chambers 130 and 140,respectively. A flooding medium, as described above, may be used to flowone or more filter aids onto the filter support.

Adding the one or more filter aids to the filter support may be carriedout in any order. In some embodiments, the at least one filter aid maybe added at the same time, sequentially, alternately, or in any relevantorder thereto. For example, as shown in FIG. 1B, only the filter aidstarch 132 is added on to the filter supports to form the pre-coatinglayers 124 and 125.

In some embodiments, adding the filter aid to the filter support forms alayer (also referred to herein as pre-coat layer layer) of the filteraid on the filter support. The pre-coat layer prevents high pressurebuild-up during filtration and prevents blockage. In some embodiments,the pre-coat layer may be uniformly distributed over the filter support.

The filter chamber may be divided into an unfiltered chamber 192, inwhich the media containing cells and/or filter aids is introduced, andfiltered chamber 194, into which the filtered medium is discharged andoptionally recirculated. For example, as shown in FIG. 1B, the pre-coatlayers 124 and 125 are formed on the filter supports 120 and 122,respectively, in the unfiltered chamber 192.

In some embodiments, the filter aid is added to the filter support atbetween about 25 g/m² -about 12000 g/m², about 50 g/m²- about 6000 g/m²,about 100 g/m² - about 3000 g/m², about 200 g/m² - about 1500 g/m², orabout 400 g/m² - about 750 g/m².

Adding a Plurality of Cells

The methods disclosed herein include adding a plurality of cells to thefilter aid and on to the filter support. In some embodiments, aplurality of cells of the same cell type may be added. In someembodiments, a plurality of cells of different cell types may be added.The plurality of cells may be added in any relevant order (e.g.simultaneously, alternately, periodically, etc.) or time interval (e.g.hourly, daily, weekly, etc.) to create a desired structure, porosity,and or functionality within the filter cake and final product.

Any cell type may be added, as described in the above section. In someembodiments, the cells may be metazoan cells. In some embodiments, thecells are selected from the group consisting of skeletal muscle cells,stem cells, pluripotent stem cells, embryonic stem cells, inducedpluripotent stem cells, fibroblasts, myoblasts, somatic cells,extraembryonic cells, myocytes, satellite cells, side population cells,muscle derived stem cells, mesenchymal stem cells, myogenic pericytes,mesoangioblasts, and adipocytes.

In some embodiments the cells exist in co-culture in the filter cake.For example, co-cultures of fibroblasts (F) and myoblasts (M) at a ratioof between about 95F:5M to about 5F:95M may be added. In anotherexample, co-cultures of adipocytes (A) and myoblasts (M) at a ratio ofbetween about 95A:5M to about 5A:95M may be added. In some embodiments,the cells are from a species of poultry, game, aquatic, or livestock. Insome embodiments, the cells are selected from the group consisting ofGallus gallus, Meleagris gallopavo, Anas platyrhynchos, Bos taurus, Susscrofa, Ovis aries, Salmo salar, Thunnus thynnus, Gadus morhua, Homarusamericanus, Litopenaeus setiferus, Oncorhynchus mykiss, and Oreochromisniloticus. In some embodiments, the cells are from a plurality of aspecies and may be added in combination, in sequence, or somecombination thereof.

Adding the plurality of cells may be carried out by any suitable method.In some embodiments, the plurality of cells is added by flowing mediacontaining the plurality of cells. For example, as shown in FIG. 1C,myoblasts 152 and fibroblasts 162 may be suspended in DMEM cell culturemedia and stored or multiplied further in cell tanks 150 and 160,respectively. The cell culture media containing the fibroblast flows onto the pre-coat layers, displacing the flooding medium via outlet 190.The fibroblasts are now embedded into the pre-coat layers on the filtersupports, forming the filter cakes 126 and 128 respectively.

Adding the plurality of cells may be carried out with or withoutaddition of one or more filter aids, in any order and any time interval.In some embodiments one cell type and one filter aid may be added to thefilter support simultaneously. For example, as shown in FIG. 1C, thefilter aid cellulose 142 may be added along with the fibroblast 162solution. In some embodiments, more than one cell type and more than onefilter support may be added to the filter support simultaneously. Forexample, as shown in FIG. 1D, the filter aids cellulose 142 and starch132 may be added along with myoblasts 152 and fibroblasts 162 to thefilter supports simultaneously.

Adding the plurality of cells may be carried out in any suitable order.In some embodiments, the plurality of cells may be added at the sametime, sequentially, alternately, or in any relevant order and any timeinterval. In some embodiments only one cell type is added. For example,as shown in FIG. 1C, only fibroblast cells 162 are added, along with thecellulose filter aid 142, to the filter supports. In some embodiments,more than one cell type is added to the filter support simultaneously.For example, as shown in FIG. 1D, both myoblasts 152 and fibroblasts 162are added to the filter cakes 126 and 128 simultaneously.

Growing the Cells into a Cell Biomass in the Filter Cake

The methods disclosed herein include growing cells into a cell biomassin the filter cake. The filter cake comprises a plurality of cells andfilter aids. In some embodiments, the filter cake is located on thefilter support. In some embodiments, the filter cake is formed on asurface of the filter support facing the unfiltered chamber. Forexample, as shown in FIG. 1D, the filter cakes 126 and 128 are locatedin the unfiltered chamber 192.

Any number or density of cells may be cultured in the filter cake. Insome embodiments, the initial density of cells in the media is betweenabout 0.05x10⁶ cells/ml - about 2000x10⁶ cells/ml, about 0.5x10⁶cells/ml - about 200x10⁶ cells/ml, about 5x10⁶ cells/ml - about 20x10⁶cells/ml, or about 5x10⁶ cells/ml - about 10x10⁶ cells/ml.

In some embodiments, culturing the cells in the filter cake includesperfusion by media (e.g. cell culture media) containing nutrients andoxygen necessary for cell growth. As described above, the continuousperfusion of nutrient rich promotes cell-cell interaction,cross-linking, and fusion of the cells in the filter cake, enabling theassembly and formation of one or more layers of thick cell biomass.

Compressing and Decompressing the Filter Cake

In some embodiments, culturing the cells in the filter cake includescompressing the filter cake. For example, in FIG. 1E the arrows 460 showthe compression and decompression of the filter cakes 126 and 128.Compressible filter aids used to create the filter cake enable changingand adapting the filter cake behavior. As discussed above, periodiccompression and decompression of the filter cake enables flow ofnutrient rich media and oxygen into all the layers of developing cellsand tissues. The compressibility of the filter cake also enables theoperator to gain information about the growth rate and adjust theporosity of the filter cake during the process. For instance, if astable, constant circulation throughout the cell-enriched filter cake isimplemented, a certain differential pressure between the unfiltered side(high pressure side) and filtered side (low pressure side), and hence adegree of filter cake compression will result. If this flow ismaintained constantly the differential pressure will increase over time,as the cells grow, divide, and fuse and thereby hinder the fluid frompenetrating more and more. In order to avoid clogging, the in-feedpressure and/or the flow rate can be reduced, leading to a relaxation ofthe cake, enabling the media to flow through the channels and pores, andcompensating the degree of clogging. The pressure/flow adjustment hencecan be correlated to the meat growth.

In some embodiments, the filter cake may be compressed by pressurevariations. In some embodiments, the pressure between the inlet and theoutlet is between about 0.1 bar - about 5.0 bar. Based on the increasedpressure difference of the filtered and unfiltered chambers, and themetabolic byproducts, conclusions can be drawn about cell proliferation,cell division, and cross-linking intensity to influence the process asneeded. By changing the flow rates to maintain a steady pressuredifference, clogging can be avoided. Alternating the pressures will leadto compression and decompression of the filter, allowing channels toform and supplying the cells in deeper layers with nutrient and oxygenenriched media.

In some embodiments, the filter cake may be compressed by flow rateand/or flow direction variations. In some embodiments, the flow ratebetween the inlet and the outlet is between about 1 L/min - about 1000L/min. In some embodiments, the flow may be from top to bottom of thefilter chamber, from bottom to top of the filter chamber, from bothsides, and/or in crossflow mode. For example, as shown in FIG. 1B, theflow may enter the filter chamber from inlet 112 and exit via outlets114 and 116. The flow rates and/or the flow directions may be variedperiodically.

Culturing may be carried out at temperature ranges suitable for cellgrowth, such as between about 10° C. to about 45° C. during growth. Asdiscussed above, temperature-controlled environments or heatingelements, such as the heat exchanger 170 may be used to set a suitabletemperature, such as about 37° C. In some embodiments, the product iscooled below 10° C. before filtration by actively cooling it and/orusing cold wash buffers.

Harvesting

In some embodiments, the method may include harvesting the cultivatedcells or the cell biomass produced therefrom, from the filter cake.After the cell biomass produced from the cultivated cells has reached adesired state (e.g. degree of growth, cell divisions, cross-linking,fiber formations, protein content, and/or overall mass), a complete orpartial degradation of filter aids can optionally be induced in thefilter chamber itself, for example by adding enzymes (which, forexample, degrade starch and/or cellulose), adding acids or bases, and/orimplementing temperature embodiments. In some embodiments, the tissuemay be obtained by physical methods (e.g. centrifugation,gravity-assisted settling), chemical methods, enzymatic methods,sedimentation, concentration, flocculation, and the like.

In some embodiments, the method may include harvesting the cultivatedcells and/or the cell biomass produced therefrom, from the filter cake,without the need to degrade or otherwise remove the filter aid. Forexample, filter cakes comprising one or more edible filter aids, thusobviating additional steps to remove or degrade filter aids. In someembodiments, the filter cake is edible. In other embodiments, the filtercake may comprise filter aids that are edible by the cell or degraded bythe cell’s secretions, whereby the filter aids are consumed, degraded,or both as the cell biomass grows.

In some embodiments, a washing medium may be used alternatively oradditionally and, if necessary, circulated for a certain period of timein order to influence the organoleptic properties (sensory and visual)of the cell biomass. For example, the washing medium may be a slightlysalty aqueous solution or a washing solution with a coloring effect(e.g. by adding juice from red beet).

In some embodiments, harvesting is carried out under sterile conditions.In some embodiments, the product is cooled down prior to harvest. Thefilter may be emptied and blown through with gas (air) to dry theproduct. In some embodiments, such as press filters, a pressing step(e.g. membrane compression) can be implemented during the process, tode-wett the filter cake, and/or to the product.

Methods for Establishing Perfusion Through a Growing Cell Biomass

Provided herein are methods for establishing perfusion through a growingcell biomass. In some embodiments, the method comprises seeding cells onan at least partially compressible filter aid, compressing the cells,the at least partially compressible filter aid, or both, flowing mediathrough the at least partially compressible filter aid to grow the cellsinto a cell biomass, whereby growth reduces perfusion over time,decompressing the cells, the at least partially compressible filter aid,or both, to increase perfusion of media.

In some embodiments, the cell biomass is compressed by a fluid pressureor flow of the media. In some embodiments, the cell culture mediaprovides nutrients and oxygenation to promote cell growth. In someembodiments, the cell biomass is decompressed with a relief valve, alower pressure media flow, flow reduction of the media, or somecombination thereof. In some embodiments, the cell biomass, itssubstrate, or both are decompressed proportionally to the growth of thecell mass, whereby perfusion is stabilized. In some embodiments, thecell biomass, the at least partially compressible filter aid, or bothcycle between a compressed state and a decompressed state at a pulsingfrequency.

In some embodiments, the pulsing frequency is about 1 second, about 10seconds, about 30 seconds, about 50 seconds, about 1 minute, about 2minutes, about 4 minutes, about 6 minutes, about 8 minutes, about 10minutes, about 12 minutes, about 14 minutes, about 16 minutes, about 18minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 2hours, about 4 hours, about 8 hours, about 16 hours, about 24 hours, orabout 48 hours. In some embodiments, the pulsing frequency is betweenabout 1 second and about 48 hours. In some embodiments, the pulsingfrequency is between about 20 seconds and about 20 minutes.

Provided herein is another exemplary filter cake-based system 200 (FIG.2A) for cultivating cells and cell biomass, and methods thereof.

FIG. 2A shows a zoomed in section of a filter chamber 210, a filtersupport 214 (e.g. a filter-candle, a filter-mash, a filter-cloth, or afilter-sieve), with the unfiltered chamber 212 on the left side of thefilter support 214, and the filtered chamber 216 on the right side ofthe filter support 214. The main flow direction here is from left toright, shown by black arrows 420 in FIG. 2A. The unfiltered chamber 212will typically have a higher pressure, because the surface and pores ofthe filter support will block and restrict flow, which will lead topressure losses. This difference in pressure is increased when thefilter support contains small or fine pores.

In some embodiments, the filter support 214 is flooded and sterilized,and the pre-coating step is carried out. FIG. 2B illustrates theintroduction of pre-coating agents, such as filter aids into the fluidstream. In this example, pre-coating agents such as compressible filteraids 218 (such as fungi fibers or cellulose) and a small increment ofincompressible filter aids 220 (such as native starch kernels) areintroduced into the fluid stream, wherein they flow towards the filtersupport 214. In one example, a combination of about 90% to about 95%compressible filter aids and about 10% to about 5% incompressible filteraids provides a favorable compressibility profile. In some embodiments,the median particle size of the pre-coating filter aids are larger thanlater added filter aids to enable optimal filtration.

FIG. 2C shows the pre-coat layer 222. Its degree of compression andhence the porosity of the filter cake are dependent on the materialsused and the differential pressure between unfiltered chamber 212 andfiltered chamber 216 (flow speed dependent). Faster flows lead to ahigher degree of compression and a higher difference between theunfiltered chamber and filtered chamber. For instance, finer particlesand a higher-pressure differential both lead to a more compressed andless porous filter cake, while large particles and a lower pressuredifferential lead to a more relaxed (or decompressed) and more porousfilter cake.

FIG. 2D illustrates the introduction of a first cell type 224 into thefluid stream (such as the various exemplary media described above),which also contains filter aids 218 and 220, and optionally other finerfilter aid compositions. The fluid stream directs the first cell type224 and the additional filter aids towards the pre-coat layer 222.

FIG. 2E illustrates the first cell type 224 embedded between bothcompressible particles 218 and incompressible particles 220, to form afirst layer of the filter cake 226. In this example, the first cell type224 is partially embedded in the filter aids of the initial filter cakeand fully embedded within the filter aids that were flowedsimultaneously with the first cells. In one example, cells are seeded ina combination of about 90% to about 95% compressible filter aids andabout 10% to about 5% incompressible filter aids.

FIG. 2F illustrates an example where a second cell type 228, along withadditional filter aids, is introduced into the fluid to create anadditional functional layer. Alternatively, co-cultures or single celltypes may be used. Although the figures illustrate pauses between eachof the individual dosing steps, in some embodiments, these processes maybe done continuously. In some embodiments, a constant differentialpressure between unfiltered chamber and filtered chamber is maintainedduring this process to ensure a constant homogeneous porosity of thecake. Since the filter cake resistance increases with increasingthickness, the flow speed (and hence amount) typically must be reducedto maintain the desired constant differential pressure.

FIG. 2G illustrates the second cell type, along with its filter aids,forming a second layer 230 of the filter cake. The second cell type maybe comprised of any cell type as disclosed herein, (such as muscularcells, fat cells, skeletal cells, or structural cells) which can bealternated and/or mixed in any order as desired. In this example, thefilter cake is comprised of a bottom layer of only filter aids (222), amiddle layer (226) of a first type of cell mixed with filter aids, and atop layer of a second type of cell mixed with filter aids (230). As thefilter cake is thickened and the cells grow, pressure may build in theunfiltered chamber, which may lead to a compression of the filter cake,a reduction in porosity, and a reduction in flow through the filtercake.

FIG. 2H illustrates the addition of a third cell type 232 along withfilter aids into the fluid, wherein they are directed towards the filtercake.

FIG. 2I illustrates a filter cake 234 layered with cells and filter aidsmany times over. This process can be repeated until the unfilteredchamber is filled to a high degree. Besides the number of cells, theamount and type of filter aids which are dosed into the stream, and theflow speed can be varied in any of the methods herein.

After the dosage of cells and filter aids is completed, nutrient andoxygen enriched cell culture medium is flown through the filter cake topromote cell growth and fusion of cells into cell biomass 236 (FIG. 2J).

FIG. 2K illustrates an example of a filter cake compressed by high flow238 (thick arrows). High flow leads to more cell-cell-contact andenables efficient cell fusion and tissue formation. Additionally, theactive compression caused by the onset of high flow forces cells, cellculture media, and oxygen to penetrate deeply into the filter cake. Onthe other hand, static compression may be associated with a decrease inporosity, whereby perfusion of culture media and oxygen becomesrestricted, which may increase the risk of cell death if prolonged. Thiscan be followed by one or multiple compression phases to supply thecells closer to the filter support with oxygen and nutrient enrichedmedium and remove undesired components. The cell biomass thickness,robustness and cell biomass will increase during these processes.

FIG. 2L illustrates an example of a filter cake 240 decompressed by lowflow, e.g. a relaxation phase. In this example, the compressed filtercake initially under high flow is subsequently exposed to a low flow togenerate a decompressed filter cake 240. In such instances, thetransition of the filter cake from a compressed state to a decompressedstate may provide several advantages to the cells within the filtercake. As the filter cake actively expands into a decompressed state,pores open throughout the filter cake and fresh cell culture media ispulled deeply and rapidly into the filter cake. A static low pressuresystem may maintain the shape and size of the pores, at leasttemporarily, formed during the decompression process, and thereby mayestablish stable perfusion to cells deep within the filter cake.However, as the cells grow and expand, the pores may become occluded,thereby necessitating another compression and decompression cycle. Thebiomass and degree of growth and fusion of the cell biomass may bedetermined by analyzing the biomass consumption rates, the decrease offlow speed if a constant differential pressure is maintained, and/or theincrease of pressure difference during a constant flow.

Once the desired biomass and degree of growth/fusion is achieved, thegrown cell biomass may be washed. In the context of biomass productionfor the generation of a cell-based meat product or other applicationswhere relevant, the washing step may adjust flavor, color and otherorganoleptic, chemical, or physical properties. FIG. 2M illustrates anadditional, optional step, where fluids 242 are added to degrade partsof the filter aid and enrich the cells per cake volume 244. Degradationand enrichment of the filter aid may be achieved with enzyme enrichedand/or tempered fluid compositions.

As shown in FIG. 2N, by stopping or reducing the flow speed ordisplacing the fluid with gas enables harvest of the cell biomassproduct 246. FIG. 2N illustrates the harvest of a grown cell mass. Inthis example, the grown cell biomass is harvested by reducing the flowspeed, thereby removing the pressure holding the grown cell biomassagainst the filter support and allowing gravity to overcome frictionresulting in the cell biomass falling off. Alternatively, the grown cellbiomass may be collected by displacing the flow of fluid with a flow ofgas, which causes the grown cell biomass to fall off.

Provided herein is yet another exemplary filter cake-based system 300(FIG. 3A) for cultivating cells and cell biomass, and methods thereof.FIGS. 3A-3M illustrate an alternative filtration approach comprising adouble-sided, vertical, and membrane press filter. This enables theoption to change flow and compression directions, which enablesobtainment of thicker filter cakes and/or lower moisture content in thecell biomass product.

FIG. 3A illustrates an exemplary system 300 comprising a filter chamber302, containing an unfiltered chamber 310, two filter supports 312 and314 (such as filter cloth), two filtered chambers 315 and 316, twoflexible structured membranes 318 and 320, and two membrane-blow-upchambers 322 and 324. The two membrane blow-up chambers 322 and 324 canbe used to inflate the membranes and reduce the unfiltered chambervolume.

As shown in FIG. 3B, after cleaning, sterilization, and flooding, afluid containing fibrous compressible filter aid 326 and a smallincrement of a less compressible filter aid 328 is introduced (indicatedby the central thick arrow 430) into the unfiltered chamber to form thepre-coat. The filtered fluid exits the filter chamber, behind the filtersupports, from the filtered chambers (indicated by the thin arrows 440at the top and bottom corners to the right and left side of the filterchamber 302).

FIG. 3C shows the pre-coat layers 330 and 332 covering both the filtersupports 314 and 312, respectively. Next, a first cell type 334 issubsequently added into the fluid stream, along with edible filter aids(FIG. 3D). As shown in FIG. 3E, the cells are embedded into the filteraids, forming the first layer of filter cakes 336 and 338 respectively.Next, a second cell type 340 is added, as shown in FIG. 3F. This resultsin additional layers being added to the filter cakes 341 and 342 (FIG.3G). Further, as shown in FIG. 3H the addition of other cell types 344can be repeated and/or alternated as desired. The process can berepeated until the unfiltered chamber is fully filled, for example 346in FIG. 3I, whereby the two initial filter cakes of the unfilteredchamber are merged into a single, thicker filter cake.

After the unfiltered chamber is filled, the flow direction can bechanged (FIG. 3J). As a result, the nutrient and oxygen enriched mediais introduced from behind the left filter support 312 (shown by arrows400 at the top and bottom of the left side of the filtered chamber 302).The fluid penetrates the porous cell biomass 348 and exits on the rightfilter support 314 (shown by arrows 410 at the top and bottom of theright side of the filtered chamber 302). Because a thick layer can leadto starvation or intoxication of cells further away from the supply,this flow direction can be alternated, and the media can also flow fromright to left. This design hence enables the creation of thicker filtercakes (e.g. essentially twice as thick) in which cells grow, multiply,fuse, and form thick cell biomass.

In some embodiments, to impact the porosity and cell-cell-contact, oneor both membranes can be inflated or deflated, as shown in FIG. 3K.Inflating the membrane blow-up chamber 322 may be achieved by using gasand/or liquid 350. Manipulating the unfiltered chamber volume(periodically or just once) can be beneficial during pre-coating,filling, cell growth, and or discharge. It helps, for instance, topulsate fluid in deep layers of the filter cake and may help open flowpaths for fluid that were otherwise closed by the growth of the growingcell mass. The inflation and deflation side can thereby be varied. Inthe example shown by FIG. 3K, the compression of the left side isaugmented by flow from the left side, whereby the compression helps theflow penetrate deeper into the filter cake.

The membrane and blow up chamber enable the filter cake to be compressedbefore it is emptied, for instance, to reduce the final moisture contentof the cell biomass product. FIG. 3L shows that both membrane blow-upchambers 322 and 324 are inflated, and a gas 354 is introduced tocompress and de-wet the filter cake. The fluid is discharged via thefilter inlet 358, or alternatively via one of the filter chambers sides.Subsequently, as shown in FIG. 3M, the blow-up chambers are deflated(indicated by arrows 450), so that the filter cake 356 can fall off andbe harvested when the unfiltered chamber is opened.

Applications

The systems and methods described herein may be used to cultivate cellsand/or cell biomass therefrom for a variety of applications. In someembodiments, the cells and/or cell biomass are cultivated into meatproducts intended for human or non-human consumption. In someembodiments, the cells and/or cell biomass are cultivated intocell-based meat (also known as in vitro produced cell-based meat, cellculture-based meat, in vitro meat, cultured meat, lab-grown meat, orclean meat).

In some embodiments, the cells are cultivated into a tissue intended fortherapeutic purposes. Examples of therapeutic use include cultivation ofstem cells, hepatocytes, 3D cell culture, cell spheroids, nodules,organoids, bio-fabrication, bio-engineering, and the like.

The foregoing description, for purposes of explanation, used specificnomenclature to provide a thorough understanding of the invention.However, it will be apparent to one skilled in the art that specificdetails are not required in order to practice the invention. Thus, theforegoing descriptions of specific embodiments of the invention arepresented for purposes of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formsdisclosed; obviously, many modifications and embodiments are possible inview of the above teachings. The embodiments were chosen and describedin order to explain the principles of the invention and its practicalapplications. These thereby enable others skilled in the art to utilizethe invention and various embodiments with various modifications as aresuited to the particular use contemplated. It is intended that thefollowing claims and their equivalents define the scope of theinvention.

Exemplary Embodiments

Some embodiments of this disclosure relate to Embodiment I, as follows:

Embodiment I-1. A method for cultivating cells and cell biomass, themethod comprising:

-   a) providing a filter support;-   b) adding at least one filter aid to the filter support;-   c) adding a plurality of cells to the filter aid, wherein the cells    and the filter aid together comprise a filter cake; and-   d) culturing the cells in the filter cake, wherein the filter cake    is perfused with media.

Embodiment I-2. The method as in embodiment I-1, wherein the filtersupport is selected from the group consisting of a horizontal filter,vertical filter, plate filter, plate press filter, mash filter, clothfilter, and candle filter.

Embodiment I-3. The method as in any one of embodiments I-1 to I-3,wherein the filter aid is selected from the group consisting ofcellulose, starch, diatomaceous earth, perlites, active charcoal, plantfibers, fungal mycella, algae, and organic fiber.

Embodiment I-4. The method of embodiment I-3, wherein the organic fibercomprises a length of between about 1 µm - 20 µm, 20 µm - 50 µm, 50 µm -80 µm, 80 µm - 110 µm, 110 µm -140 µm, 140 µm - 170 µm, 170 µm - 200 µm,200 µm - 230 µm, 230 µm - 260 µm, 260 µm -290 µm, or 290 µm - 320 µm.

Embodiment I-5. The method of embodiment I-3, wherein the organic fiberhas a fiver titer of between about 0.01 dtex - 120 dtex, 0.05 dtex - 60dtex, 0.1 ldtex - 30 dtex, 0.2 dtex - 15 dtex, or 0.5 dtex - 5 dtex.

Embodiment I-6. The method as in any one of embodiments I-1 to I-5,wherein the filter aid is compressible.

Embodiment I-7. The method as in any one of embodiments I-1 to I-6,wherein the filter aid is compressible by between about 70% - 75%, 75% -80%, 80% - 85%, 85% - 90%, 90% - 95%, 95% - 98%, or 98% - 100%.

Embodiment I-8. The method as in any one of embodiments I-1 to I-7,wherein the filter aid is hollow.

Embodiment I-9. The method as in any one of embodiments I-1 to 1-8,wherein the filter aid is edible.

Embodiment I-10. The method as in any one of embodiments I-1 to I-9,wherein the filter aid is degradable.

Embodiment I-11. The method as in any one of embodiments I-1 to I-10,further comprising compressing the filter cake by varying the flow rateof the media.

Embodiment I-12. The method as in any one of embodiments I-1 to I-11,further comprising compressing the filter cake by varying the pressure.

Embodiment I-13. The method as in any one of embodiments I-1 to I-12,wherein the filter aid is added to the filter support at between about25 g/m² - 12000 g/m², 50 g/m² - 6000 g/m², 100 g/m² - 3000 g/m², 200g/m² - 1500 g/m², or 400 g/m² - 750g/m².

Embodiment I-14. The method as in any one of embodiments I-1 to I-13,wherein the density of cells in the filter cake is between about0.05x10⁶ cells/ml - 2000x10⁶ cells/ml, 0.5x10⁶ cells/ml - 200x10⁶cells/ml, 5x10⁶ cells/ml - 20x10⁶ cells/ml, or 5x10⁶ cells/ml - 10x10⁶cells/ml.

Embodiment I-15. The method as in any one of embodiments I-1 to I-14,wherein the temperature is maintained at between about 10° C. - about45° C.

Embodiment I-16. The method as in any one of embodiments I-1 to I-15,wherein the cells are metazoan cells.

Embodiment I-17. The method as in any one of embodiments 1-1 to I-16,wherein the cells are selected from the group consisting of skeletalmuscle cells, stem cells, pluripotent stem cells, embryonic stem cells,induced pluripotent stem cells, fibroblasts, myoblasts, somatic cells,extraembryonic cells, myocytes, satellite cells, side population cells,muscle derived stem cells, mesenchymal stem cells, myogenic pericytes,mesoangioblasts, and adipocytes.

Embodiment I-18. The method as in any one of embodiments I-1 to I-17,wherein the cells are co-cultures of fibroblast and myoblasts at a ratioof between about 95F:5M to 5F:95M.

Embodiment I-19. The method as in any one of embodiments I-1 to I-18,wherein the cells are from a species of poultry, game, aquatic, orlivestock.

Embodiment I-20. The method as in any one of embodiments I-1 to I-19wherein the cells are selected from the group consisting of Gallusgallus, Meleagris gallopavo, Anas platyrhynchos, Bos taurus, Sus scrofa,Ovis aries, Salmo salar, Thunnus thynnus, Gadus morhua, Homarusamericanus, Litopenaeus setiferus, Oncorhynchus mykiss, and Oreochromisniloticus.

Embodiment I-21. The method as in any one of embodiments 1-1 to I-20,wherein adding the at least one filter aid comprises flowing mediacontaining the at least one filter aid.

Embodiment I-22. The method as in any one of embodiments 1-1 to I-21,wherein the at least one filter aid is added at the same time,sequentially, alternately, or in any relevant order thereto.

Embodiment I-23. The method as in any one of embodiments I-1 to I-22,wherein adding the plurality of cells comprises flowing media containingthe plurality of cells.

Embodiment I-24. The method as in any one of embodiments I-1 to I-23,wherein the plurality of cells are added at the same time, sequentially,alternately, or in any relevant order thereto.

Embodiment I-25. The method as in any one of embodiments I-1 to I-24,wherein the filter cake is edible.

Embodiment I-26. The method as in any one of embodiments 1-1 to I-25,further comprising harvesting the cells or the tissues producedtherefrom, from the filter cake.

Embodiment I-27. The method as in any one of embodiments I-1 to I-26,wherein the media comprises GRAS-ingredients, optionally onlyGRAS-ingredients.

Embodiment I-28. The method as in any one of embodiments I-1 to I-27,wherein the cells are cultivated into a tissue intended for humanconsumption.

Embodiment I-29. The method as in any one of embodiments I-1 to I-28,wherein the cells are cultivated into a tissue intended for therapeuticpurposes.

Embodiment I-30. A system for cultivating cells and cell biomass, thesystem comprising:

-   a) a filter chamber comprising at least one inlet and at least one    outlet;-   b) at least one filter support located within the filter chamber;    and-   c) a filter cake located on the filter support, wherein the filter    cake comprises at least one filter aid and a plurality of cells.

Embodiment I-31. The system of embodiment I-30, wherein the filter cakecomprises at least two different cell types.

Embodiment I-32. The system as in any one of embodiments I-30 to I-31,wherein the cells are selected from metazoan cells.

Embodiment I-33. The system as in any one of embodiments I-30 to I-32,wherein the cells are selected from the group consisting of skeletalmuscle cells, stem cells, pluripotent stem cells, embryonic stem cells,induced pluripotent stem cells, fibroblasts, myoblasts, somatic cells,extraembryonic cells, myocytes, satellite cells, side population cells,muscle derived stem cells, mesenchymal stem cells, myogenic pericytes,mesoangioblasts, and adipocytes.

Embodiment I-34. The system as in any one of embodiments I-30 to I-33,wherein the cells are co-cultures of fibroblast and myoblasts at a ratioof between about 95F:5M to 5F:95M.

Embodiment I-35. The system as in any one of embodiments I-30 to I-34,wherein the cells are from a species of poultry, game, aquatic, orlivestock.

Embodiment I-36. The system as in any one of embodiments I-30 to I-35,wherein the cells are selected from the group consisting of Gallusgallus, Meleagris gallopavo, Anas platyrhynchos, Bos taurus, Sus scrofa,Ovis aries, Salmo salar, Thunnus thynnus, Gadus morhua, Homarusamericanus, Litopenaeus setiferus, Oncorhynchus mykiss, and Oreochromisniloticus.

Embodiment I-37. The system as in any one of embodiments I-30 to I-36,wherein the filter chamber is in fluid communication with at least onereservoir containing cells.

Embodiment I-38. The system as in any one of embodiments I-30 to I-37,wherein the filter chamber is in fluid communication with at least onereservoir containing the filter aid.

Embodiment I-39. The system as in any one of embodiments I-30 to I-38,wherein the temperature of the filter chamber is maintained at betweenabout 10° C. - about 45° C.

Embodiment I-40. The system as in any one of embodiments I-30 to I-39,wherein the flow rate between the inlet and the outlet is between about1 L/min - 1000 L/min.

Embodiment I-41. The system as in any one of embodiments I-30 to I-40,wherein the pressure between the inlet and the outlet is between about0.1 bar - 5.0 bar.

Embodiment I-42. The system as in any one of embodiments I-30 to I-41,wherein the filter aid is added to the filter support at between about25 g/m² - 12000 g/m², 50 g/m² - 6000 g/m², 100 g/m² - 3000 g/m², 200g/m² - 1500 g/m², or 400 g/m² - 750 g/m².

Embodiment I-43. The system as in any one of embodiments I-30 to I-42,wherein the filter chamber has a size of between about 5 liter - 25,000liters.

Embodiment I-44. The system as in any one of embodiments I-30 to I-43,wherein the filter support has surface area of between about 1 meter² -2000 meter².

Embodiment I-45. The system as in any one of embodiments I-30 to I-44,wherein the filter cake comprises two cell types in alternating layers.

Embodiment I-46. The method as in any one of embodiments I-1 to I-29,wherein the filter cake comprises two cell types in alternating layers.

Embodiment II-1. A method for optimizing the cultivation of cells andcell biomass, the method comprising:

-   a) providing a filter support;-   b) adding at least one filter aid to the filter support, wherein the    at least one filter aid is compressible;-   c) adding a plurality of cells to the filter aid, wherein the cells    and the filter aid together comprise a filter cake;-   d) compressing the filter cake; and-   e) culturing the cells in the filter cake.

Embodiment II-2. The method of embodiment II-1, comprising harvestingthe cells and the tissues produced therefrom.

Embodiment II-3. The method of embodiment II-1, wherein a meat productis thereby generated.

Embodiment II-4. The method of embodiment II-1, comprising compressingthe filter cake by varying one or more of flow rate and pressure.

Embodiment II-5. The method of embodiment II-1, wherein the filter cakeis edible.

Embodiment II-6. The method of embodiment II-1, wherein the at least onefilter aid is compressible.

Embodiment II-7. The method of embodiment II-1 ,wherein the at least onefilter aid is edible.

Embodiment II-8. The method of embodiment II-1, wherein the at least onefilter aid is selected from the group consisting of cellulose, starch,diatomaceous earth, perlites, active charcoal, plant fibers, fungalmycella, algae, and organic fibers.

Embodiment II-9. The method of embodiment II-1, wherein the organicfibers comprise a length of between about 1 µm - 20 µm, 20 µm - 50 µm,50 µm - 80 µm, 80 µm - 110 µm, 110 µm -140 µm, 140 µm - 170 µm, 170 µm -200 µm, 200 µm - 230 µm, 230 µm - 260 µm, 260 µm -290 µm, or 290 µm -320 µm.

Embodiment II-10. The method of embodiment II-1, wherein the organicfiber has a fiver titer of between about 0.01 dtex - 120 dtex, 0.05dtex - 60 dtex, 0.1 dtex - 30 dtex, 0.2 dtex - 15 dtex, or 0.5 dtex - 5detx.

Embodiment II-11. The method of embodiment II-1, wherein the at leastone filter aid is added to the filter support at between about 25 g/m² -12000 g/m², 50 g/m² - 6000 g/m², 100 g/m² -3000 g/m², 200 g/m² - 1500g/m², or 400 g/m² - 750 g/m².

Embodiment 11-12. The method of embodiment II-1, wherein the filtersupport is selected from the group consisting of a horizontal filter,vertical filter, plate filter, plate press filter, mash filter, clothfilter, and a candle filter.

Embodiment 11-13. The method of embodiment II-1, wherein the temperatureis maintained at between about 10° C. to about 45° C.

Embodiment 11-14. The method of embodiment II-1, wherein the cells arefrom a species of poultry, game, aquatic, or livestock.

Embodiment 11-15. The method of embodiment II-1, wherein the cells areselected from the group consisting of Gallus gallus, Meleagrisgallopavo, Anas platyrhynchos, Bos taurus, Sus scrofa, Ovis aries, Salmosalar, Thunnus thynnus, Gadus morhua, Homarus americanus, Litopenaeussetiferus, Oncorhynchus mykiss, and Oreochromis niloticus.

Embodiment 11-16. The method of embodiment II-1, wherein adding the atleast one filter aid comprises flowing media containing the at least onefilter aid.

Embodiment 11-17. The method of embodiment II-1, wherein adding theplurality of cells comprises flowing media containing the plurality ofcells.

Embodiment 11-18. A system for cultivating cells and cell biomass, thesystem comprising:

-   a) a filter chamber comprising at least one inlet and at least one    outlet;-   b) at least one filter support located within the filter chamber;    and-   c) a filter cake located on the filter support, wherein the filter    cake is compressible, and comprises at least one filter aid and a    plurality of cells.

Embodiment 11-19. The system of embodiment 11-18, wherein the filterchamber is in fluid communication with at least one reservoir containingcells.

Embodiment II-20. The system of embodiment 11-18, wherein the filterchamber is in fluid communication with at least one reservoir containingthe filter aid.

Embodiment III-1. A method for optimizing the cultivation of cells andcell biomass, the method comprising:

-   a) providing a filter support;-   b) adding at least one filter aid to the filter support;-   c) adding a plurality of cells to the filter aid, wherein the cells    and the filter aid together comprise a filter cake; and-   d) growing the cells into a cell biomass in the filter cake, wherein    the filter cake is at least partially compressible.

Embodiment III-2. The method of embodiment III-1, wherein the cellbiomass is a meat product.

Embodiment III-3. The method of embodiment III-1, comprising compressingor decompressing the filter cake by varying one or more of flow rate andpressure.

Embodiment III-4. The method of embodiment III-1, wherein the filtercake is edible.

Embodiment III-5. The method of embodiment III-1, wherein the at leastone filter aid is at least partially compressible.

Embodiment III-6. The method of embodiment III-1, wherein the at leastone filter aid is edible or degradable.

Embodiment III-7. The method of embodiment III-1, wherein the filter aidcomprises at least one compressible filter aid and one non-compressiblefilter aid.

Embodiment III-8. The method of embodiment III-1, wherein the organicfibers comprise a length of between about 1 µm - 20 µm, 20 µm - 50 µm,50 µm - 80 µm, 80 µm - 110 µm, 110 µm -140 µm, 140 µm - 170 µm, 170 µm -200 µm, 200 µm - 230 µm, 230 µm - 260 µm, 260 µm -290 µm, or 290 µm -320 µm.

Embodiment III-9. The method of embodiment III-1, wherein the organicfiber has a fiver titer of between about 0.01 dtex - 120 dtex, 0.05dtex - 60 dtex, 0.1 dtex - 30 dtex, 0.2 dtex - 15 dtex, or 0.5 dtex - 5detx.

Embodiment III-10. The method of embodiment III-1, wherein the at leastone filter aid is added to the filter support at between about 25 g/m² -12000 g/m², 50 g/m² - 6000 g/m², 100 g/m² -3000 g/m², 200 g/m² - 1500g/m², or 400 g/m² - 750 g/m².

Embodiment III-11. The method of embodiment III-1, wherein the filtersupport is contained in a horizontal filter, vertical filter, platefilter, plate press filter, mash filter, cloth filter, mass filter, orcandle filter.

Embodiment III-12. The method of embodiment III-1, wherein thetemperature is maintained at between about 10° C. to about 45° C.

Embodiment III-13. The method of embodiment III-1, wherein the cells arefrom a species of poultry, game, aquatic, or livestock.

Embodiment III-14. A method for establishing perfusion through a growingcell biomass, the method comprising:

-   a) seeding cells on an at least partially compressible filter aid;-   b) compressing the cells, the at least partially compressible filter    aid, or both;-   c) flowing media through the at least partially compressible filter    aid to grow the cells into a cell biomass, whereby growth reduces    perfusion over time; and-   d) decompressing the cells, the at least partially compressible    filter aid, or both, to increase perfusion of media.

Embodiment III-15. The method of embodiment III-14, wherein the cellbiomass is compressed by a fluid pressure or flow of the media.

Embodiment III-16. The method of embodiment III-14, wherein the cellculture media provides nutrients and oxygenation to promote cell growth.

Embodiment III-17. The method of embodiment III-14, wherein the cellbiomass is decompressed with a relief valve, a lower pressure mediaflow, flow reduction of the media, or some combination thereof.

Embodiment III-18. The method of embodiment III-14, wherein the cellbiomass, its substrate, or both are decompressed proportionally to thegrowth of the cell mass, whereby perfusion is stabilized.

Embodiment III-19. The method of embodiment III-14, wherein the cellbiomass, the at least partially compressible filter aid, or both cyclebetween a compressed state and a decompressed state at a pulsingfrequency.

Embodiment III-20. The method of embodiment III-19, wherein the pulsingfrequency is between 20 seconds and 20 minutes.

1. A method comprising: providing a filter support; adding at least onefilter aid to the filter support; adding a plurality of cells to the atleast one filter aid, wherein the plurality of cells and the at leastone filter aid together comprise a filter cake; and culturing theplurality of cells in the filter cake by perfusing the filter cake withmedia.
 2. The method as in claim 1, wherein the filter support isselected from the group consisting of a horizontal filter, verticalfilter, plate filter, plate press filter, mash filter, cloth filter, andcandle filter.
 3. The method as in claim 1, wherein the at least onefilter aid is selected from the group consisting of cellulose, starch,diatomaceous earth, perlites, active charcoal, plant fibers, fungalmycella, algae, and organic fiber.
 4. The method as in claim 1, whereinthe at least one filter aid is hollow.
 5. The method as in claim 1,wherein the at least one filter aid is edible.
 6. The method as in claim1, wherein the at least one filter aid is degradable.
 7. The method asin claim 1, further comprising compressing the filter cake by varying aflow rate of the media through the filter cake.
 8. The method as inclaim 1, further comprising compressing the filter cake by varying apressure of a chamber containing the filter cake.
 9. The method as inclaim 1, wherein adding the plurality of cells comprises flowing themedia containing the plurality of cells through the at least one filteraid.
 10. The method as in claim 1, further comprising harvesting theplurality of cells, or tissues produced therefrom, from the filter cake.11. The method as in claim 1, wherein adding the plurality of cells tothe at least one filter aid comprises adding two cell types inalternating layers.
 12. A method comprising: providing a filter support;adding at least one filter aid to the filter support; adding a pluralityof cells to the at least one filter aid, wherein the plurality of cellsand the at least one filter aid together comprise a filter cake; andgrowing the plurality of cells into a cell biomass in the filter cake.13. The method of 12, wherein the cell biomass is a meat product. 14.The method of 12, further comprising compressing or decompressing thefilter cake by varying one or more of flow rate and pressure of mediathrough the filter cake.
 15. The method of 12, wherein the at least onefilter aid is edible or degradable.
 16. The method of 12, whereinproviding the filter support comprises providing the filter support in ahorizontal filter, vertical filter, plate filter, plate press filter,mash filter, cloth filter, mass filter, or candle filter.
 17. The methodof 12, further comprising maintaining a temperature of the filter cakeat between about 10° C. to about 45° C. during growth of the pluralityof cells.
 18. The method of claim 12, wherein the filter cake is atleast partially compressible.
 19. The method of claim 12, whereingrowing the plurality of cells into the cell biomass in the filter cakecomprises providing nutrients and oxygen to the plurality of cells inthe filter cake enabling one or more of cell growth, cell division, cellfusion, or cross-linking.
 20. The method of claim 12, wherein growingthe plurality of cells into the cell biomass in the filter cake comprisecausing fusion of myoblasts into multi-nucleated myotubes within thefilter cake.